Since antibodies have extremely specific molecular recognition ability and high avidity and can easily be produced, with high probability, for a target molecule to be analyzed, antibodies have been utilized as extremely useful research reagents in many laboratories and have been practically utilized in a wide range of applications from diagnostic reagents to pharmaceutical agents for several decades. In many cases, the targets of an antibody are proteins although antibodies have also been utilized for the highly-sensitive detection of target low-molecular-weight compounds such as a certain kind of drug and environmental pollutant.
After the completion of human genome analysis, recently, the expression status and the function of several tens of thousands of gene products are being actively studied in mammalian cells with a focus on human. Several tens of thousands to several hundred thousands of proteins are mixed in tissues and cells. To examine the expression status and localization of a particular one of these proteins, an antibody is essential. In the studies of differentiation and tissue regeneration as well as of cancer cells and stem cells, an antibody has become an indispensable tool as a differentiation marker-detection means for cell sorting in which cells with different functions are fractionated from the same population and for understanding the control of differentiation. Moreover, antibodies are frequently used in basic research of correlation between a particular protein molecule and a certain disease. These studies lead to the development of diagnostic reagents. Antibody drugs are developed from studies on the neutralization of a particular protein molecule and the therapeutic effect on a disease. As described above, antibodies are utilized in a wide range of applications, from basic research to direct practical applications.
To obtain antibodies, animals are usually immunized by antigens. The antigens may be natural or artificial. For natural antigens, a purified target protein or a partially-purified or unpurified mixture containing a target protein is used. On the other hand, major types of artificially prepared antigens are classified into the following two types: (i) a recombinant protein produced by expressing a gene coding the target protein or a fragment thereof in a suitable host and (ii) a synthetic peptide of an amino acid sequence of a portion of the target protein.
The term “peptide” as used herein refers to a peptide with the length of 3 to about 40 amino acids.
If a synthetic peptide is used as an antigen, the time and effort required for antigen preparation can advantageously significantly be reduced due to fewer impurities, as compared to the case of purifying a target protein from natural cells or tissues or the case of utilizing recombinant gene expression. Since the amino acid sequence information is easily available at present with the development of DNA sequencing techniques, immunization using a synthetic peptide as an antigen is frequently utilized. Another advantage of using a synthetic peptide as an antigen is that a particular region of protein can be selected.
The length of a synthetic peptide frequently used as an antigen is usually around 10 to 25 amino acids. To cause an immune reaction, an antigen must bind to the B cell and the class II T cell at the same time (Non Patent Literature 1, P.72). When immunization is performed with a typical immunization schedule, i.e., one administration per one to two weeks, the antigen must remain in the body for more than a certain period of time after administration to an animal. Although the antigen must have at least a certain molecular weight to satisfy these requirements, a peptide generally has a small molecular weight and rapidly metabolized after administration and therefore is not directly used as an antigen.
The immunization with a peptide is disadvantageous in that even if an antibody specifically binding to the peptide is obtained, the antibody does not necessarily have good reactivity to the original protein containing the sequence thereof (Non Patent Literature 1). In this regard, when a peptide is used as an antigen, the probability of acquisition of an intended antibody is lower than when the target protein itself or a fragment thereof (with a molecular weight of about 5000 or more) is used as an antigen and, therefore, two or three different peptide antigen sequences are generally tested.
With regard to which amino acid sequence of the target protein should be selected as a peptide antigen, absolutely certain method is not yet known. As a general selection criteria, one should avoid a position likely to be glycosylated (a region including a motif of Asn-X-Thr or a region rich in Ser or Thr), and select a portion having a relatively high degree of hydrophilicity and likely to come out of a molecular surface or a site containing proline or a bent portion such as the β-turn (Non Patent Literature 1, Non Patent Literature 2).